The sodium-potassium adenosine triphosphatase (i.e. Na,K-ATPase) is responsible for controlling cellular fluid and electrolyte balance in higher eukaryotes. The Na,K-ATPase is a heterodimeric integral membrane protein consisting of a catalytic a-subunit (~1000 amino acids) and a glycosylated [unreadable]-subunit (~300 amino acids). During some physiological states, this single enzyme is responsible for utilizing nearly 40% of the cells energy. In kidney and intestinal epithelial cells the Na,K-ATPase is strictly delivered to the basolateral membrane providing directional uptake of Na and other solutes (e.g. glucose, amino acids). Recently, several laboratories have reported that in addition to solute transport, the Na,K-ATPase is a cell-surface receptor for cardiac glycoside-mediating Src signaling. Some of these alternative physiological roles of the Na,K-ATPase mandate that the enzyme be delivered to specific sub-plasma membrane pools, which raises as yet unaddressed questions pertaining to Na,K-ATP maturation and membrane targeting. The planned experiments in this proposal will focus on the cell physiological roles of Na,K-ATPase, focusing on a novel intracellular physiological function. Our previous work revealed for the first time that the Na pump is completely functional once assembled within the endoplasmic reticulum. However, the potential physiological role of this intracellular activity remained a mystery. Now recent evidence has emerged demonstrating that the Na/Ca exchanger plays a role in nuclear calcium homeostasis in some cells which immediately suggests that there must be a sodium concentration gradient across the nuclear envelope to drive this secondary active transporter. In this application, we focus on demonstrating that this intracellular [Na+] gradient is established and maintained by the Na,K-ATPase functioning intracellularly. Indeed, we provide preliminary data showing that the Na,K-ATPase resides within the nuclear envelope in HEK-293 cells and is catalytically active in this membrane. The specific aims addressed in this R21 proposal will provide important new information about Na,K-ATPase and provide the foundation for a new avenue of investigation regarding intracellular membrane transport to regulate nucleoplasmic Ca2+ signaling. These aims are: 1) Demonstrate that the Na,K-ATPase participates in nuclear Na+ and Ca2+ transport, 2) Identify proteins that retain Na,K-ATPase within the nuclear envelope, and 3) Determine the tissue distribution of nuclear Na,K-ATPase targeting. We will combine strategies from molecular biology, cell physiology, biochemistry, electrophysiology and confocal microscopy to accomplish our scientific goals. The Na,K-ATPase is the pharmacological target for cardiac glycosides, a widely used therapy for congestive heart failure. Considering the mounting evidence that cellular distribution of Na,K-ATPase plays a key role in its physiology, the work proposed here will be crucial to understanding and resolving the etiology of clinically relevant problems. Specifically, dysregulation of the Na,K-ATPase has been attributed to hypertension, congestive heart failure, familial hemiplegic migraine, epileptogenesis, and polycystic kidney disease. PUBLIC HEALTH RELEVANCE: The Na,K-ATPase is an essential transport system and the site of action of digitalis, the most widely used therapy to treat patients with congestive heart failure. Prospects for improved therapies for cardiac function, as well as improving impaired renal function, will be greatly aided when we have a better understanding of the regulation of the activity of the Na,K-ATPase in cell membranes and by elucidating the mechanisms by which cells properly deliver this vital enzyme to specific subcellular locations.